Methods for antibody engineering

ABSTRACT

The invention provides a method for identifying positions of an antibody that can be modified without significantly reducing the binding activity of the antibody. In many embodiments, the method involves identifying a substitutable position in a parent antibody by comparing its amino acid sequence to the amino acid sequences of a number of related antibodies that each bind to the same antigen as the parent antibody. The amino acid at the substitutable position may be substituted for a different amino acid without significantly affecting the activity of the antibody. The subject methods may be employed to change the amino acid sequence of a CDR without significantly reducing the affinity of the antibody of the antibody, in humanization methods, or in other antibody engineering methods. The invention finds use in a variety of therapeutic, diagnostic and research applications.

FIELD OF THE INVENTION

The field of this invention is antibodies, particularly methods forengineering, e.g., humanizing, monoclonal antibodies.

BACKGROUND OF THE INVENTION

Because of their ability to target virtually any molecule with exquisitespecificity, monoclonal antibodies have the potential to become one ofthe main therapeutic agents of the future. Though this potential wasrecognized several years ago, however the first attempts to fulfill thepotential were disappointing because monoclonal antibodies used intherapy elicit a strong immune response in patients (Schroff, 1985Cancer. Res. 45:879-85, Shawler. J Immunol 1985 135:1530-5), even at lowdoses (Dillman, Cancer Biother. 1994 9:17-28). Scientists predict thathuman antibodies would not cause such adverse immune responses. However,no suitable methods exist for producing human monoclonal antibodies.Alternative technologies to make human antibodies using, for example,phage display and transgenic animals have been developed more recentlybut are not widely used for therapeutic purposes.

The immunogenicity of antibodies depends on many factors, including themethod of administration, the number of injections, the dosage, thenature of the conjugation, the specific fragment utilized, the state ofaggregation and the nature of the antigen (e.g., Kuus-Reichel, Clin.Diagn. Lab. Immunol. 1994 1:365-72). Many or most of these factors canbe manipulated in order to decrease an immune response. However, if theoriginal antibody sequence is recognized as “dangerous” or “foreign”,the chances are that sooner or later a strong immune response willprevent the use of that antibody in therapy.

In order to decrease these responses, efforts have been made to replaceas much as possible of the non-human sequence of an antibody with humansequences using recombinant DNA technology. Towards this end, chimericantibodies containing human antibody light chain and heavy chainconstant domains that are joined to mouse antibody variable light chainand heavy chain domains have been employed. Chimeric antibodies stillcontain a large number of non-human amino acid sequences in the variableregions and, as such, a significant immune response may be mountedagainst such antibodies. CDR grafting is another humanization techniquein which the antigen binding portions or “complementarity determiningregions” (CDRs) of monoclonal antibodies are grafted by recombinant DNAtechnologies into the DNA sequences encoding the framework (i.e. thenon-CDR region) of human antibody heavy and light chains. One technicalproblem of CDR grafted antibodies is that they usually show considerabledecreased affinity. To restore increase the affinity of CDR graftedantibodies, certain original key framework residues (e.g., residues thatare thought to be involved in determining the conformation of the CDRs)are reintroduced into the CDR grafted antibody. Using a differenthumanization approach, Roguska devised a “resurfacing” strategy formouse antibodies where only exposed residues that are different toexposed residues of a human antibody are substituted.

However, although antibodies humanized by the above methods can showreduced immunogenicity in human patients (Moreland, Arthritis Rheum 199336:307-18) many humanized antibodies are still highly immunogenic to alarge proportion of patients. This is thought to be because the CDRsthemselves are immunogenic (Ritter, Cancer Res 2001 61:6851-9; Welt,Clin Cancer Res 2003 9:1338-46).

All of the methods described above require that the CDR regions of thenon-human antibody remain unchanged during the humanization process inorder to maintain antibody specificity and affinity. However, sincenon-human CDR regions are themselves immunogenic in humans, methods forhumanizing the CDR regions of a non-human antibody without significantlyreducing the binding activity of the antibody are highly desirable. Theidentification of suitable methods for humanizing the CDR regions of anon-human antibody has been a daunting, if not impossible, task for themedical and research community.

Accordingly, there is an ongoing need for improved methods for makingnon-human antibodies that are less immunogenic in humans and othermammalian hosts. In particular, there is a need for humanization methodsthat reduce the immunogenicity of CDR regions of a non-human antibody inhumans. The present invention meets this, and other, needs.

LITERATURE

References of interest include: U.S. Pat. Nos. 6,331,415 B1, 5,225,539,6,342,587, 4,816,567, 5,639,641, 6,180,370, 5,693,762, 4,816,397,5,693,761, 5,530,101, 5,585,089, 6,329,551, and publications Morea etal., Methods 20: 267-279 (2000), Ann. Allergy Asthma Immunol. 81:105-119(1998), Rader et al., J. Biol. Chem. 276:13668-13676 (2000), Steinbergeret al., J. Bio. Chem. 275: 36073-36078 (2000), Roguska et al., Proc.Natl. Acad. Sci. 91: 969-973 (1994), Delagrave et al., Prot. Eng. 12:357-362 (1999), Rogusca et al., Prot. Eng. 9: 895-904 (1996), Knight andBecker, Cell 60: 963-970 (1990); Becker and Knight, Cell 63:987-997(1990) Popkov, J Mol Biol 325:325-35 (2003); Rader et al., Proc. Natl.Acad. Sci. 95:8910-8915; Mehr et al., J. Immunol. 172:4790-6 (2004) andDe Pascalis et al. J Imm. 2002, 169:3076-3084.

SUMMARY OF THE INVENTION

The invention provides a method for identifying positions of an antibodythat can be modified without significantly reducing the binding activityof the antibody. In many embodiments, the method involves identifying asubstitutable position in a parent antibody by comparing its amino acidsequence to the amino acid sequences of a number of related antibodiesthat each bind to the same antigen and epitope as the parent antibody.The amino acid at the substitutable position may be substituted for adifferent amino acid without significantly affecting the activity of theantibody. The subject methods may be employed to change the amino acidsequence of a CDR without significantly reducing the affinity of theantibody of the antibody, in humanization methods, or in other antibodyengineering methods. The invention finds use in a variety oftherapeutic, diagnostic and research applications.

These and other advantages and features of the invention will becomeapparent to those persons skilled in the art upon reading the details ofthe invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram illustrating one embodiment of the invention.

FIG. 2 is an amino acid sequence alignment illustrating an exemplarymethod by which substitutable positions within a CDR region may beidentified. From top to bottom, the amino acid sequences shown in FIG. 2are SEQ ID NOS: 1-11.

FIG. 3 is two panels showing an exemplary amino acid sequence alignmentillustrating one aspect of an exemplary method by which the CDR regionsof an antibody may be humanized. From top to bottom, the amino acidsequences shown in FIG. 3 are SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO:14; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 15.

FIG. 4 is an exemplary amino acid sequence alignment. From top tobottom, the amino acid sequences shown in FIG. 4 are SEQ ID NOS: 16-25.Beta strand positions are shown at the top. The adopted numbering system(see Chothia, below) is shown near the top. The following positions areindicated: c: are CDR contacts; i: are at the interface of VK/VH; b: areinternal buried residues (see Padlan, below) and C are CDR residues. Thesequences are labeled according to convention.

FIG. 5 shows the amino acid sequence of 20 exemplary VH3 regions of arabbit antibodies. From top to bottom, the amino acid sequences shown inFIG. 5 are SEQ ID NOS: 26-45.

FIG. 6 is an exemplary amino acid sequence alignment illustrating oneaspect of an exemplary method by which a rabbit antibody may behumanized. From top to bottom, the amino acid sequences shown in FIG. 6are SEQ ID NOS: 46-48.

FIG. 7 shows an an exemplary amino acid sequence alignment illustratinghow a consensus sequence for an antibody can be made.

DEFINITIONS

Before the present subject invention is described further, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantibody” includes a plurality of such antibodies and reference to “aframework region” includes reference to one or more framework regionsand equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. These terms are well understood by those in the field, and referto a protein consisting of one or more polypeptides that specificallybinds an antigen. One form of antibody constitutes the basic structuralunit of an antibody. This form is a tetramer and consists of twoidentical pairs of antibody chains, each pair having one light and oneheavy chain. In each pair, the light and heavy chain variable regionsare together responsible for binding to an antigen, and the constantregions are responsible for the antibody effector functions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen, and monoclonal antibodies.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), andHunkapiller and Hood, Nature, 323, 15-16 (1986),).

An immunoglobulin light or heavy chain variable region consists of a“framework” region (FR) interrupted by three hypervariable regions, alsocalled “complementarity determining regions” or “CDRs”. The extent ofthe framework region and CDRs have been precisely defined (see,“Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S.Department of Health and Human Services, (1991)). The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs. The CDRs are primarilyresponsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain geneshave been constructed, typically by genetic engineering, from antibodyvariable and constant region genes belonging to different species. Forexample, the variable segments of the genes from a mouse monoclonalantibody may be joined to human constant segments, such as gamma 1 andgamma 3. An example of a therapeutic chimeric antibody is a hybridprotein composed of the variable or antigen-binding domain from a rabbitantibody and the constant or effector domain from a human antibody(e.g., the anti-Tac chimeric antibody made by the cells of A.T.C.C.deposit Accession No. CRL 9688), although other mammalian species may beused. As used herein, the term “humanized antibody” or “humanizedimmunoglobulin” refers to an non-human (e.g., mouse or rabbit) antibodycontaining one or more amino acids (in a framework region, a constantregion or a CDR, for example) that have been substituted with acorrespondingly positioned amino acid from a human antibody. In general,humanized antibodies produce a reduced immune response in a human host,as compared to a non-humanized version of the same antibody.

It is understood that the humanized antibodies designed and produced bythe present method may have additional conservative amino acidsubstitutions which have substantially no effect on antigen binding orother antibody functions. By conservative substitutions is intendedcombinations such as those from the following groups: gly, ala; val,ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr. Aminoacids that are not present in the same group are “substantiallydifferent” amino acids.

The term “specific binding” refers to the ability of an antibody topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. In certain embodiments, aspecific binding interaction will discriminate between desirable andundesirable analytes in a sample, in some embodiments more than about 10to 100-fold or more (e.g., more than about 1000- or 10.000-fold).

In certain embodiments, the affinity between a capture agent and analytewhen they are specifically bound in a capture agent/analyte complex ischaracterized by a K_(D) (dissociation constant) of less than 10⁻⁶M,less than 10⁻⁷M, less than 10⁻⁸ M, less than 10⁻⁹ M, less than 10⁻⁹ M,less than 10⁻¹¹ M, or less than about 10⁻¹² M or less.

An amino acid residue that is in “close contact”, “close proximity” or“in close proximity to” another amino acid residue is an amino acidresidue that is has a side chain that is close to, i.e., within 7, 6, 5or 4 Angstroms of, a side chain of another amino acid. For example, anamino acid that are proximal to a CDR is a non-CDR amino acid that has aside chain that is close to a side chain of an amino acid in a CDR.

A “variable region” of a heavy or light antibody chain is an N-terminalmature domain of the chains. All domains, CDRs and residue numbers areassigned on the basis of sequence alignments and structural knowledge.Identification and numbering of framework and CDR residues is asdescribed in by Chothia and others (Chothia Structural determinants inthe sequences of immunoglobulin variable domain. J Mol Biol 1998;278:457-79).

VH is the variable domain of an antibody heavy chain. VL is the variabledomain of an antibody light chain, which could be of the kappa (K) or ofthe lambda isotype. K-1 antibodies have the kappa-1 isotype whereas K-2antibodies have the kappa-2 isotype and VL is the variable lambda lightchain.

A “buried residue” is an amino acid residue whose side chain has lessthan 50% relative solvent accessibility, which is calculated as thepercentage of the solvent accessibility relative to that of the sameresidue, X, placed in an extended GGXGG peptide. Methods for calculatingsolvent accessibility are well known in the art (Connolly 1983 J. appl.Crystallogr, 16, 548-558).

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like. Polypeptides may be of any size, and theterm “peptide” refers to polypeptides that are 8-50 residues (e.g., 8-20residues) in length.

As used herein the term “isolated,” when used in the context of anisolated antibody, refers to an antibody of interest that is at least60% free, at least 75% free, at least 90% free, at least 95% free, atleast 98% free, and even at least 99% free from other components withwhich the antibody is associated with prior to purification.

The terms “treatment” “treating” and the like are used herein to referto any treatment of any disease or condition in a mammal, e.g.particularly a human or a mouse, and includes: a) preventing a disease,condition, or symptom of a disease or condition from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; b) inhibiting a disease, condition, or symptomof a disease or condition, e.g., arresting its development and/ordelaying its onset or manifestation in the patient; and/or c) relievinga disease, condition, or symptom of a disease or condition, e.g.,causing regression of the condition or disease and/or its symptoms.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans. Other subjects mayinclude cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses,and so on.

“Corresponding amino acids”, as will be described in greater detailbelow, are amino acid residues that are at an identical position (i.e.,they lie across from each other) when two or more amino acid sequencesare aligned. Methods for aligning and numbering antibody sequences areset forth in great detail in Chothia, supra, Kabat supra, and others. Asis known in the art (see, e.g. Kabat 1991 Sequences of Proteins ofImmunological Interest, DHHS, Washington, D.C.), sometimes one, two orthree gaps and/or insertions of up to one, two, three or four residues,or up to about 15 residues (particularly in the L3 and H3 CDRs) may bemade to one or both of the amino acids of an antibody in order toaccomplish an alignment.

A “natural” antibody is an antibody in which the heavy and lightimmunoglobulins of the antibody have been naturally selected by theimmune system of a multi-cellular organism, as opposed to unnaturallypaired antibodies made by e.g. phage display. As such, the subjectparental antibodies do not usually contain any viral (e.g.,bacteriophage M13)-derived sequences. Spleen, lymph nodes and bonemarrow are examples of tissues that produce natural antibodies.

A “substitutable position”, as will be described in greater detailbelow, is a particular position of an antibody that may be substitutedby different amino acids without significantly decreasing the bindingactivity of the antibody. Methods for identifying substitutablepositions, and how they may be substituted, are described in muchgreater detail below. A substitutable positions may also be referred toas “variation tolerant position”.

A “parent” antibody, as will be described in greater detail below, is anantibody is the target of amino acid substitutions. In certainembodiments, amino acids may be “donated” by a “donor” antibody to theparent antibody to produce an altered antibody.

“Related antibodies”, as will be described in greater detail below, areantibodies that have a similar sequence and produced by cells that havea common B cell ancestor. Such a B cell ancestor contains a genomehaving a rearranged light chain VJC region and a rearranged heavy chainVDJC region, and produces an antibody that has not yet undergoneaffinity maturation. “Naïve” or “virgin” B cells present in spleentissue, are exemplary B cell common ancestors. Related antibodies bindto the same epitope of an antigen and are typically very similar insequence, particularly in their L3 and H3 CDRs. Both the H3 and L3 CDRsof related antibodies have an identical length and a near identicalsequence (i.e., differ by 0, 1 or 2 residues). Related antibodies arerelated via a common antibody ancestor, the antibody produced in thenaïve B cell ancestor. The term “related antibodies” is not intended todescribe a group of antibodies that do not have a common antibodyancestor produced by a B-cell.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention provides a method for identifying positions of an antibodythat can be modified without significantly reducing the binding activityof the antibody. In many embodiments, the method involves identifying asubstitutable position in a parent antibody by comparing its amino acidsequence to the amino acid sequences of a number of related antibodiesthat each bind to the same antigen and epitope as the parent antibody.The amino acid at the substitutable position may be substituted for adifferent amino acid without significantly affecting the activity of theantibody. The subject methods may be employed to change the amino acidsequence of a CDR without significantly reducing the affinity of theantibody of the antibody, in humanization methods, or in other antibodyengineering methods. The invention finds use in a variety oftherapeutic, diagnostic and research applications.

In further describing the subject invention, methods of identifyingvariation-tolerant positions are discussed first, followed by adescription of various protocols in which those methods find use.

Methods for Identifying a Variation-Tolerant Position of an Antibody

As mentioned above, the invention provides a method for identifying avariation-tolerant, i.e., substitutable, position of an antibody. Oncesuch a position is identified, the amino acid at that position may besubstituted for a different amino acid without significantly decreasingthe binding activity of the antibody. The subject method is particularlyemployable in methods in which it is desirable to identify substitutableresidues in regions of an antibody that would otherwise be thought ofbeing essential for antigen binding. For example, the subject methodsmay be employed to identify substitutable positions in a CDR region ofan antibody. In particular embodiments, the subject methods may beemployed to identify a substitutable position in a CDR region of anantibody that is to be humanized. Once identified, the amino acid atthat position can be substituted for a “human” amino acid (e.g., anamino acid that occupy the equivalent position of a human germlineantibody that has a sequence similar the antibody to be humanized).Accordingly, the subject method find particular use in humanizationmethods, although, as will be described in greater detail below, thesubject methods may be readily employed in a wide variety of antibodyengineering methods.

In very general terms and with reference to FIG. 1, the subject methodsinvolve immunizing an antibody-producing animal with an antigen 2, andobtaining the amino acid sequence of several monoclonal antibodies thatbind to that antigen 4. The amino acid sequences of these antibodies arethen compared (e.g., by aligning those sequences), and the antibodiesare classified according to their similarity to each other to identifyrelated groups of antibodies 6. The antibodies within each group ofrelated antibodies generally share a common ancestor antibody, and haveevolved from that ancestor antibody via somatic hypermutation, geneconversion and other cellular mutation-producing mechanisms that occurduring affinity maturation and the final stages of B-cell development.Once groups of related antibodies have been established, the amino acidsequences of the antibodies within a group can be compared to identifysubstitutable positions 8. A substitutable position of an individualantibody may be identified by virtue of the fact that the identity ofthe amino acid at that position varies between the individual antibodiesof a group of related antibodies. Once identified, the amino acid at thesubstitutable position of an individual antibody can be substituted fora different amino acid without significantly decreasing the affinity ofthe antibody 10. Since antibodies containing amino acid substitutions atthese substitutable positions were originally produced and effectivelytested by the immune system of the initial immunized animal,substitution at those positions should be well tolerated by theantibody. In particular embodiments, an amino acid substitution may be ahumanizing substitutions (i.e., a substitution that make the amino acidsequence more similar to that of a human antibody) 12, a directedsubstitution (e.g., a substitution that make the amino acid sequence ofan antibody more similar to that of a related antibody) 14, a randomsubstitution (e.g., a substitution with any of the naturally-occurringamino acids) or a conservative substitution (e.g., a substitution withan amino acid having biochemical properties similar to that beingsubstituted).

As mentioned above, the subject method involves immunizing a suitableanimal with an antigen, and obtaining the amino acid sequences ofseveral antigen-reactive antibodies from that animal. The antibody aminoacid sequences are usually obtained by sequencing cDNAs encoding theheavy and light chains of those antibodies. The cDNAs are obtained fromantibody-producing cells of the animal.

Any suitable animal, e.g., a warm-blooded animal, in particular a mammalsuch as a rabbit, mouse, rat, camel, sheep, cow or pig or a bird such asa chicken or turkey, may be immunized with a selected antigen using anyof the techniques well known in the art suitable for generating animmune response. Procedures for immunizing animals are well known in theart, and are described in Harlow (Antibodies: A Laboratory Manual, FirstEdition (1988) Cold Spring Harbor, N.Y.) and Weir (Handbook ofExperimental Immunology Vol 4, Blackwell Scientific Publishers, Oxford,England, 1986). In particular embodiments, a rabbit having an undefinedor defined genotype may be employed.

Within the context of the present invention, the phrase “selectedantigen” includes any substance to which an antibody may be made,including, among others, polypeptides (including peptides),carbohydrates, inorganic or organic molecules, transition state analogsthat resemble intermediates in an enzymatic process, nucleic acids,cells, including cancer cells, cell extracts, pathogens, includingliving or attenuated viruses, bacteria and the like. As will beappreciated by one of ordinary skill in the art, antigens which are oflow immunogenicity may be accompanied with an adjuvant or hapten inorder to increase the immune response (for example, complete orincomplete Freund's adjuvant) or with a carrier such as keyhole limpethemocyanin (KLH). Suitable antigens include extracellularly-exposedfragments of Her2, GD2, EGF-R, CEA, CD52, CD20, Lym-1, CD6, complementactivating receptor (CAR), EGP40, VEGF, tumor-associated glycoproteinTAG-72 AFP (alpha-fetoprotein), BLyS (TNF and APOL—related ligand),CAl25 (carcinoma antigen 125), CEA (carcinoembrionic antigen), CD2(T-cell surface antigen), CD3 (heteromultimer associated with the TCR),CD4, CD11 a (integrin alpha-L), CD14 (monocyte differentiation antigen),CD20, CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25(IL-2 receptor alpha chain), CD30 (cytokine receptor), CD33 (myeloidcell surface antigen), CD40 (tumor necrosis factor receptor), CD44v6(mediates adhesion of leukocytes), CD52 (CAMPATH-1), CD80 (costimulatorfor CD28 and CTLA-4), complement component C5, CTLA, EGFR, eotaxin(cytokine A11), HER2/neu, HLA-DR, HLA-DR10, HLA ClassII, IgE, GPiib/iiia(integrin), Integrin aVβ3, Integrins a4β1 and a4β7, Integrin β2,IFN-gamma, IL-1β, 1L-4, IL-5, IL-6R (IL6 receptor), IL-12, IL-15, KDR(VEGFR-2), lewisy, mesothelin, MUC1, MUC18, NCAM (neural cell adhesionmolecule), oncofetal fibronectin, PDGFI3R (Beta platelet-derived growthfactor receptor), PMSA, renal carcinoma antigen G250, RSV, E-Selectin,TGFbeta1, TGFbeta2, TNFalpha, TRAIL-R1, VAP-1 (vascular adhesionprotein 1) or VEGF, or the like.

In many embodiments, a peptide having the amino acid sequencecorresponding to a portion of an extracellular domain of one of theabove-listed proteins is employed as an antigen.

Once a suitable animal has been immunized and an immune response againstthe antigen has been established by the animal, antibody producing cellsfrom the animal are screened to identify cells that produce antibodieshaving a desired activity. In many embodiments, these methods may employhybridoma technology. In other embodiments, however, the methods mayemploy flow cytometry (FACS) of cell populations obtained from rabbitspleen, bone marrow, lymph node, plasma or other lymph organs, e.g.,through incubating the cells with labeled anti-rabbit IgG and sortingthe labeled cells using a FACSVantage SE cell sorter (Becton-Dickinson,San Jose, Calif.).

In many embodiments nucleic acids encoding the VH and VL domains of anantibody are isolated from an antibody-producing hybridoma cell. Inorder to produce antibody-producing hybridoma lines, an animal isimmunized with an antigen and once a specific immune response of therabbit has been established, cells from the spleen of the immunizedanimal are fused with a suitable immortal cell (e.g., NIH 3T3, DT-40 or240E cell, etc.; Spieker-Polet et al, Proc. Natl. Acad. Sci. 92:9348-9352, 1995) to produce hybridoma cells. Supernatants from thesehybridoma cells are screened for antibody secretion by enzyme-linkedimmunosorbent assay (ELISA) and positive clones secreting monoclonalantibodies specific for the antigen can be selected and expandedaccording to standard procedures (Harlow et al., Antibodies: ALaboratory Manual, First Edition (1988) Cold spring Harbor, N.Y.; andSpieker-Polet et al., supra). Suitable monoclonal antibodies may befurther selected in the basis of binding activity, including its bindingspecificity, binding affinity, binding avidity, a blocking activity orany other activity that causes an effect (e.g. promoting or inhibiting acellular phenotype, e.g., cell growth, cell proliferation, cellmigration, cell viability (e.g., apoptotis), cell differentiation, celladherence, cell shape changes (e.g., tubular cell formation), complementdependant cytotoxicity CDC, antibody-dependent cell-mediatedcytotoxicity ADCC, receptor activation, gene expression changes, changesin post-translational modification (e.g., phosphorylatoin), changes inprotein targeting (e.g., NFKB localization etc.), etc., or inhibition ofreceptor multimerization (e.g., dimer or trimerization) orreceptor-ligand interactions).

Antibody-encoding nucleic acids are isolated from these cells usingstandard molecular biology techniques such as polymerase chain reaction(PCR) or reverse transcription PCR (RT-PCR) (Ausubel, et al, ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook,et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989)Cold Spring Harbor, N.Y.). In particular embodiments, sequences encodingat least the variable regions of the heavy and light chains areamplified from cDNA using techniques well known in the art, such asPolymerase Chain Reaction (PCR). See Mullis, U.S. Pat. No. 4,683,195;Mullis et al., U.S. Pat. No. 4,683,195; Polymerase Chain Reaction:Current Communication in Molecular Biology, Cold Springs Harbor Press,Cold Spring Harbor, N.Y., 1989. Briefly, cDNA segments encoding thevariable domain of the antibody are exponentially amplified byperforming sequential reactions with a DNA polymerase. The reaction isprimed by a 5′ and a 3′ DNA primer. In some embodiments, the 3′antisense primer corresponding to a DNA sequence in the constant (orjoining) region of the immunoglobulin chain and the 5′ primer (or panelof related primers) corresponding to a DNA sequence in the variableregion of the immunoglobulin chain. This combination of oligonucleotideprimers has been used in the PCR amplification of murine immunoglobulincDNAs of unknown sequence (see Sastry et at., Proc Natl. Acad. Sci.86:5728-5732, 1989 and Orlandi et al., Proc. Natl. Acad. Sci.86:3833-3837, 1989). Alternatively, an “anchored polymerase chainreaction” may be performed (see Loh et al., Science 243:217-220, 1989).In this procedure, the first strand cDNA is primed with a 3′ DNA primeras above, and a poly(dG tail) is then added to the 3′ end of the strandwith terminal deoxynucleotidyl transferase. The product is thenamplified by PCR using the specific 3′ DNA primer and anotheroligonucleotide consisting of a poly(dC) tail attached to a sequencewith convenient restriction sites. In many embodiments, however, theentire polynucleotide encoding a heavy or light chain is amplified usingprimers spanning the start codons and stop codons of both of theimmunoglobulin cDNAs, however, depending on the amplification productsdesired, suitable primers may be used. In a representative embodiment,rabbit antibody-encoding nucleic acids can be amplified using thefollowing primers: heavy chain, 5′ end(CACCATGGAGACTGGGCTGCGCTGGCTTCTCCTGGTCGCTGTG; SEQ ID NO:49); heavychain, 3′ end (CTCCCGCTCTCCGGGTAAATGAGCGCTGTGCCGGCGA; SEQ ID NO:50);light chain kappa, 5′end (CAGGCAGGACCCAGCATGGACACGAGGGCCCCCACT; SEQ IDNO:51); and L kappa, 3′ end (TCAATAGGGGTGACTGTTAGAGCGAGACGCCTGC; SEQ IDNO:52). Suitable restriction sites and other tails may be engineeredinto the amplification oligonucleotides to facilitate cloning andfurther processing of the amplification products. Amplificationprocedures using nested primers may also be used, where such nestedprimers are well known to one of skill in the art. The variable domainsof the antibodies may be sequenced directly from PCR products, or fromcloned DNA fragments.

Accordingly an animal is immunized with an antigen, and the amino acidsequence of a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 ormore, 15 or more, 20 or more, 30 or more, 50 or more, 80 or more 100 ormore, usually up to 500 or 1000 or more) of monoclonal antibodies thatbind to that antigen are obtained. In certain embodiments, themonoclonal antibodies are obtained from the cells of a single animalimmunized with the antigen.

Once the amino acid sequences of the V_(H) and V_(L) domains of a set ofantigen-binding antibodies have been determined, the amino acids arecompared to identify a group of related antibodies that have a similarsequence. This may be done by numbering the amino acid positions of eachantibody using a suitable numbering system, such as that provided byChothia or Kabat supra. CDR and/or framework residues may be identifiedusing these methods. The numbered sequences may be aligned by eye, or byemploying an alignment program such as one of the CLUSTAL suite ofprograms (Thompson et al Nucleic Acids Research, 22:4673-4680). Thevariable regions of antibodies within a related group of antibodies haveamino acid sequences that are very similar. For example, the VH or VLdomains of antibodies within a related group of antibodies may haveamino acid sequences that are at least about 90% identical (e.g., atleast 95% or at least 98% or at least 99% identical), ignoring any gapsor insertions made to facilitate alignment of the sequences. Antibodieswithin a related group of antibodies have a VL domains that are similarto each other, as well as VH domains that are similar to each other. Inother words, in certain embodiments the VH or VL domains of twodifferent related antibodies usually contain up to about five (i.e.,one, two, three, four or five or more) amino acid differences. An aminoacid difference may be present at any position of the variable domain,including in any CDR or in any framework region. Related rabbitantibodies have H3 CDRs that are almost identical, as well as L3 CDRsthat are almost identical. In these embodiments, any two antibodies thatare related will have L3 and H3 CDRs that are each identical in lengthand have near identical sequences (i.e., that contain 0, 1 or 2 aminoacid changes). In other words the L3 CDRs of the two antibodies areidentical in length and near identical in sequence and the H3 CDRs ofthe two antibodies are identical in length and near identical insequence. Two exemplary sets of related antibodies are shown in FIG. 4,and the sequences of 20 exemplary VH3 regions of unrelated rabbitantibodies are shown for comparison.

Depending on the particular antigen used, the species and genotype ofthe animal used, and the number of antibody-encoding nucleic acidssequenced, a relatively low number (e.g., less than about 5 or 10 groupsmay be identified). In certain embodiments, only one or two groups maybe identified. The antibodies within each group display greater than 90%sequence to each other, whereas any two antibodies of any two differentgroups typically display less than 90% to each other, across the entirelength of the variable domains of the antibodies.

In order to identify a substitutable position of an antibody, the aminoacid sequence of that antibody is compared to the sequences of otherantibodies belonging to the same group as that antibody. If the identityof that amino acid varies between the different related antibodies of agroup at any particular position, that position is a substitutableposition of the antibody. In other words, a substitutable position is aposition in which the identity of the amino acid varies between therelated antibodies. Positions that contain a constant amino acid are notsubstitutable positions. This aspect of the invention may be exemplifiedwith reference to FIG. 2. FIG. 2 shows an exemplary amino acid sequencealignment of 10 different exemplary, hypothetical, antibodies that arerelated. The amino acid sequences of the framework regions (FW) of theseantibodies are omitted from FIG. 2, although the principles discussedabove and below are readily applicable to framework sequences. At eachposition the amino acid can be invariable (i.e., constant) or variable(may change) from on antibody to another. In the example shown in FIG.2, the amino acid at positions a, b, d, e, g, h, i, j, k, m, n, o, q, r,s, u, v, w, x, z and α are constant, whereas the amino acids atpositions c, f, l, p, t and y are variable. Positions c, f, l, p, t andy are substitutable (or variation tolerant) positions whereas positionsa, b, d, e, g, h, i, j, k, m, n, o, q, r, s, u, v, w, x, z and α are notsubstitutable positions.

In a further embodiment, the above method may be employed to provide aconsensus antibody sequence. In such a consensus sequence, anon-substitutable position is indicated by the amino acid present atthat position, and a substitutable position is indicated as an “X”.Depending on how the antibodies are to be employed, X may be a) anyamino acid, b) any amino acid present at that position in any of therelated antibodies in the group or a conservatively substituted variantthereof or c) any amino acid present at that position in any of therelated antibodies in the group. For example, in the example shown inFIG. 2, the antibody consensus has a sequence:RTXATXCLFQ-FW1-RXWTVXA-FW2-PSXSHTVXIT (SEQ ID NO:54), where X can be anyamino acid, any amino acid present at that position in a relatedantibody, or a conservatively substituted amino acid present at thatposition in a related antibody. Any antibody having a sequence that isencompassed by the consensus should bind to the same antigen as any ofthe related antibodies. Exemplary consensus sequences for the heavy andlight chains of three sets of related antibodies that bind to TNFα areshown in FIG. 7. The non-X amino acids are the same as those shown atthe equivalent position of the antibody sequences shown in FIG. 4. Incertain embodiments, a consensus sequence may only contain the aminoacid sequence of the CDR regions of an antibody.

Substituting an Amino Acid at a Substitutable Position

The method described above may be employed in methods of designing andmaking a variant of a parental antibody that at least maintains (i.e.maintains or increases) the antigen binding activity of the parentalantibody. Because antibodies containing substitutions at substitutablepositions have already been produced and tested by an immunized animal,substitutions at those positions can be made in the knowledge that theyshould not significantly decrease the binding activity of the antibody.In general, an antibody variant of a parental antibody has an antigenbinding affinity that is at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 100% (e.g., at least 150%, at least 200%, at least500%, at least 1000%, usually up to at least 10,000%) of the bindingaffinity of the parental antibody to a particular antigen.

As illustrated in FIG. 1, a substitutable position of a parentalantibody may be substituted by a) any of the 20 naturally occurringamino acids to produce random substitutions, b) an amino acid havingbiochemical properties similar to the amino acid already present at thesubstitutable position to produce conservative substitutions, c) anamino acid that is present at the same position in a related antibody toproduce a directed substitution, or d) an amino acid that is present atthe same position in a similar human antibody to produce a humanizingsubstitution. A substitution may be made at any part of an antibodyvariable region, including any framework region or CDR. In certainembodiments, a single substitutable amino acid may be substituted.However, in other embodiments, a plurality of substitutable amino acids(e.g., up to about 5 or 10 ore more) may be substituted. In particularembodiments, the type of substitution that can be made at eachsubstitutable position may be indicated by the types of amino acidspresent at that position in the related antibodies. For example, ifunrelated amino acids (e.g., ala, gly, cys, glu and thr) are present ata certain position of a group of related antibodies, then any amino acidcould be substituted at that position without significantly reducingbinding activity of the antibody. Similarly, if a subset of non-polaramino acids (e.g., val, ile, ala and met) are present at a certainposition of a set of related antibodies, then other non-polar aminoacids (e.g., leu) could be substituted at that position withoutsignificantly reducing binding activity of the antibody.

In any of these methods, the resultant antibody variants may be testedto confirm that any binding activities have not been significantlyreduced by substitution. Further, and as will be described in greaterdetail below, a library of variant antibodies that contain a pluralityof substituted amino acids may be produced, and screened to provide anantibody with an improved activity. For example, one or moresubstitutable positions of an antibody may be substituted by anycombination of random, conservative or directed substitutions to producea library of variants that are each individually tested to identify anantibody having an improved binding activity.

Conservative Substitutions

The amino acid at a substitutable position of an antibody may bereplaced by an amino acid having similar properties (based on size,polarity, hydrophobicity, and the like) to the amino acid to bereplaced. In other words, the amino acid at a substitutable position ofan antibody can be replaced with a different amino acid of the sameclass, where the amino acids may be classified as follows: aromatic:phe, tyr, trp; apolar: leu, val, ile, ala, met; aliphatic: ala, val,leu, ile; acidic: asp, glu; basic: his, lys, arg; polar: gln, asn, ser,thr, tyr. In certain embodiments, the amino acid at a substitutableposition of an antibody may be replaced according to the followingtable:

amino acid to be replacing replaced amino acid Ala Ser Arg Lys Asn Gln,His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Directed Substitutions

The amino acid at a substitutable position of an antibody may bereplaced by a different amino acid that is present at the same positionin a related antibody (i.e., a related antibody). For example and withreference to FIG. 2, the ala at substitutable position c in antibody 1could be replaced with a gly, cys, glu or a thr since these amino acidsare found at substitutable position c in antibodies 3, 5, 7 and 10,respectively; the met at substitutable position fin antibody 1 could bereplaced with a val or an ile, since these amino acids are found atsubstitutable position fin antibodies 4 and 8, respectively; the phe atsubstitutable position 1 in antibody 1 could be replaced with a tyr ortrp, since these amino acids are found at substitutable position 1 inantibodies 6 and 9, respectively, and so on for positions p, t and y ofantibody 1.

Humanizing Substitutions

The amino acid at a substitutable position of a parental antibody may bereplaced by a different amino acid that is present at the same positionof a human antibody. In these embodiments, the amino acid sequence ofthe variable domain of a parental antibody is usually compared to adatabase of human antibody sequences, and a human antibody that has anamino acid sequence that is similar to that of the parental antibody isselected. The amino acid sequence of the parental antibody and the humanantibody are compared (e.g., aligned), and one or more substitutableamino acids of the parental antibody are substituted by correspondinglypositioned amino acids in the human antibody. This embodiment isexemplified in the top panel of FIG. 3, where all substitutable aminoacids are substituted for their human counterpart. The bold underlinedamino acids of the humanized sequence (hmAb) indicate amino acids thathave been substituted. The bold double-underlined amino acids have notbeen substituted since the “human” amino acid was already present in theparental antibody.

In a refinement of this embodiment, the humanizing substitution may be adirected substitution in which an amino acid at a substitutable positionis substituted for an amino acid that is present in both the humanantibody and a related antibody. This embodiment is illustrated in thebottom panel of FIG. 3. In this figure, the ala at position c ofantibody 1 is substituted with a thr, where a thr is found at thatposition in both antibody 10 (as shown in FIG. 2) and a similar humanantibody. Further, the gln at position y of antibody 1 is substitutedwith a tyr, where a tyr is found at that position in both antibody 9 (asshown in FIG. 2) and a similar human antibody. Other substitutable aminoacids (i.e., those at positions f, l, p and t) are not substituted inthis embodiment since none of the related antibodies have the same aminoacid as the human antibody at this position.

In other embodiments, the substituting amino acids may be chosen asbeing less polar than the other amino acids, and therefore lessimmunogenic.

A suitable human antibody for use in these methods is identified bycomparing the heavy and light chain variable domain sequences of theparental antibody (or a consensus sequence of set of related antibodies)to a database of human antibody sequences. Typically, one of the 10 mostsimilar sequences in terms of amino acid sequence identity (either bypercent identity or P-value) will be employed as an amino acid residuedonor. In certain embodiment, one of the three most similar antibodies(e.g., the most similar) in terms of amino acid sequence identity(percent identity or P-value) to a parental antibody sequence may beused as an amino acid residue donor. The selected human antibody and theparental antibody will typically have at least about 55%, at least about65% identity, at least about 75%, at least about 80%, at least about85%, at least about 90%, or at least about 95% amino acid sequenceidentity across the entire variable domain in one or both of thesequenced chains. In certain embodiments, both the light and heavychains from the same human antibody may be used as amino acid donors. Inmost embodiments, the parental antibody is compared to human germ-lineantibody sequences.

Various antibody databases can be searched to identify the mosthomologous human antibody immunoglobulins for a given rabbitimmunoglobulin sequence. In addition to National Center forBiotechnology Information (NCBI) databases, several of the most commonlyused databases are listed below:

V BASE—Database of Human Antibody Genes: This database is maintained bythe medical research council (MRC), of Cambridge UK and is provided viathe website: www.mrc-cpe.cam.ac.uk. This database is comprehensivedirectory of all human germline variable region sequences compiled fromover a thousand published sequences, including those in the currentreleases of the Genbank and EMBL data libraries.

Kabat Database of Sequences of Proteins of Immunological Interest(Johnson, G and Wu, TT (2001) Kabat Database and its applications:future directions. Nucleic Acids Research, 29: 205-206) found at thewebsite of Northwestern University, Chicago (immuno.bme.nwu.edu).

Immunogenetics Database: Maintained by and found at the website of theEuropean Bioinformatics Institute: www.ebi.ac.uk. This database isintegrated specialized database containing nucleotide sequenceinformation of genes important in the function of the immune system. Itcollects and annotates sequences belonging to the immunoglobulinsuperfamily which are involved in immune recognition.

ABG: Germline gene directories of the mouse—a directory of mouse VH andVK germline segments, part of the webpage of the Antibody Group at theInstituto de Biotecnologia, UNAM (National Univfrsity of Mexico)

Built-in searching engines can be used to search for most similarsequences in terms of amino acid sequence homology. In the methods ofthis invention, BLAST (Altschul et al., J. Mol. Biol. 215:403-10, 1990)is performed using default parameters, including choosing the BLOSUM62matrix, an expect threshold of 10, low complexity filter off, gapsallowed, and a word size of 3.

During the subject humanization methods, one, two, three, four, five orsix or more, usually up to about 10 or more, humanizing amino acidsubstitutions are made. Non-consecutive amino acids are generallysubstituted in these methods.

The above-described methods for making humanizing substitutions in anantibody may be employed as an alternative to, in combination with, orin addition to known antibody humanization methods such as the CDRgrafting and resurfacing methods discussed in the introduction.

For example, the subject humanization methods may be incorporated intoany humanization method that requires making amino acid substitutions ina parental antibody to make it more similar to a known human antibody(see, e.g., U.S. patent application serial nos. 10/638,210 and10/637,317, both filed on Aug. 7, 2003, and other references cited inthe background, all incorporated by reference herein in their entirety).For example, many prior humanization methods are directed to identifyingparticular amino acids in a parental antibody that can be substituted bya human amino acid (i.e., the amino acid found at the same position in ahuman antibody). As a refinement of these prior methods, the instantmethods can be employed to identify which of those particular aminoacids are substitutable amino acids and are therefore variationtolerant. Since amino acid substitutions at these substitutablepositions are readily tolerated by an antibody (i.e., they don'tsignificantly decrease binding affinity), humanizing amino acidsubstitutions can be made without significantly reducing antibodyactivity. For example, only substitutable positions that are on thesurface of an antibody and not in a significant area of secondarystructure may be substituted by a human amino acid. In addition, themethod may be employed in combination with methods for removing helper Tcell epitopes from an antibody, such as the “deimmunization” methodsdescribed in published U.S. Patent No. 20030153043 and others. Forexample, only deimmunizating amino acid changes that occur atsubstitutable positions may be made. Such changes should not abolishantibody activity.

In particular embodiments, the subject methods may be employed tohumanize the CDRs of an antibody. These embodiments may be employed inaddition to other humanization methods that are directed to humanizingthe framework regions and other non-CDR regions of an antibody, forexample.

The humanization methods described above represent a significantcontribution to the antibody humanization arts because no otherhumanization method can be employed to substitute only those positionsof an antibody that are known to be tolerant to substitutions.

Further, since the instant methods effectively employ the amino acidsequences of variant antibodies that have been selected as having strongbinding activity by the immune system of the immunized animal (byaffinity maturation), substituting an amino acid at a substitutableposition of an antibody identified by the above methods often leads toan increase in binding affinity. This is particularly true of antibodiesthat have been subjected to directed substitutions, as described above.Accordingly, in general, the instant humanization methods may beemployed to humanize a parental antibody to produce a humanized antibodythat has a greater binding affinity for an antigen than the parentalantibody.

Methods of Improving Antibody Activity

In one embodiment of particular interest, the instant substitutionsmethods may be employed to improve a binding activity of a parentalantibody. As noted above, the substitutable positions identified by thesubject methods are sites that are employed to improve the bindingactivity of a progenitor antibody during affinity maturation. Thosepositions, and the amino acids present into those positions in the groupof related antibodies, were selected as increasing the affinity of anantibody to a particular antigen. By combining the individual changesmade to an antibody during affinity maturation, an antibody having anincreased affinity for an antigen may be produced. In certainembodiments, therefore, a plurality of directed substitutions may bemade in a parental antibody to increase the affinity of that antibody.For example, a parental antibody may be modified to contain the mostcommon substitution at each of the substitutable positions of a group ofrelated antibodies.

In a related method, if a sufficient number of antibodies (e.g., morethen 20 and up to about 50 or more) are sequenced, particular antibodyactivities (e.g., antibody binding affinity, antibody binding avidity,antibody binding specificity, etc.) of those antibodies can becorrelated with particular amino acid changes. This knowledge allows anantibody having a combination of selected binding activities to bedesigned and made.

Further, and as mentioned above, the identification of substitutablepositions of an antibody facilitates the production of libraries ofcandidate antibodies to be screened to identify an antibody have adesired binding activity. In one example, this method involves makingevery possible combination of amino acid substitutions (e.g., anycombination of directed, random and/or conservative substitutions forexample) at substitutable positions of an antibody to produce anantibody library that can be screened to identify an antibody having animproved properties.

Suitable methods for screening antibodies are well known in the art, andinclude but are not limited to the following:

Binding Assays

In these assays, each antibody of a subject library is tested for itsability to bind specifically to a substrate. The term “specifically” inthe context of antibody binding, refers to high avidity and/or highaffinity binding of an antibody to a specific antigen i.e., apolypeptide, or epitope. In many embodiments, the specific antigen is anantigen (or a fragment or subfraction of an antigen) used to immunizethe animal host from which the antibody-producing cells were isolated.Antibody specifically binding an antigen is stronger than binding of thesame antibody to other antigens. Antibodies which bind specifically to apolypeptide may be capable of binding other polypeptides at a weak, yetdetectable, level (e.g., 10% or less of the binding shown to thepolypeptide of interest). Such weak binding, or background binding, isreadily discernible from the specific antibody binding to a subjectpolypeptide, e.g. by use of appropriate controls. In general, specificantibodies bind to an antigen with a binding affinity with a KD of 10⁻⁷Mor less, e.g., 10⁻⁸ M or less (e.g., 10⁻⁹M or less, 10⁻¹⁰ or less, 10¹¹or less 10⁻¹² or less, or 10¹³ less, etc.). In general, an antibody witha binding affinity KD of 10⁻⁷ M or greater is not useful in that it willnot bind an antigen at a detectable level using conventional methodologycurrently used. Typically, in performing a screening assay, antibodysamples produced by a library of antibody producing host cells aredeposited onto a solid support in a way that each antibody can beidentified, e.g. with a plate number and position on the plate, oranother identifier that will allow the identification of the host cellculture that produced the antibody.

The antibodies of the invention may be screened for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, and protein A immunoassays, to name but a few. Such assaysare routine and well known in the art (see, e.g., Ausubel et al, eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally involve lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4.degree. C., adding protein Aand/or protein G sepharose beads to the cell lysate, incubating forabout an hour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads).

Western blot analysis generally involves preparation of protein samplesfollowed by electrophoresis of the protein samples in a polyacrylamidegel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), and transfer of the separated protein samples from thepolyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon.Following transfer, the membrane is blocked in blocking solution (e.g.,PBS with 3% BSA or non-fat milk), washed in washing buffer (e.g.,PBS-Tween 20), and incubated with primary antibody (the antibody ofinterest) diluted in blocking buffer. After this incubation, themembrane is washed in washing buffer, incubated with a secondaryantibody (which recognizes the primary antibody, e.g., an anti-humanantibody) conjugated to an enzymatic substrate (e.g., horseradishperoxidase or alkaline phosphatase) or radioactive molecule (e.g., 32Por 1251), and after a further wash, the presence of the antigen may bedetected. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise.

ELISAs involve preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Antibodies of the invention may be screened using immunocytochemistymethods on cells (e.g., mammalian cells, such as CHO cells) transfectedwith a vector enabling the expression of an antigen or with vector aloneusing techniques commonly known in the art. Antibodies that bind antigentransfected cells, but not vector-only transfected cells, are antigenspecific.

In certain embodiments, however, the assay is an antigen capture assay,and an array or microarray of antibodies may be employed for thispurpose. Methods for making and using microarrays of polypeptides areknown in the art (see e.g. U.S. Pat. Nos. 6,372,483, 6,352,842,6,346,416 and 6,242,266).

Inhibitor Assays

In certain embodiments, the assay measures the specific inhibition of anantibody to an interaction between a first compound and a secondcompound (e.g. two biopolymeric compounds) or specifically inhibits areaction (e.g. an enzymatic reaction). In the interaction inhibitionassay, one interaction substrate, usually a biopolymeric compound suchas a protein e.g. a receptor, may be bound to a solid support in areaction vessel. Antibody is added to the reaction vessel followed by adetectable binding partner for the substrate, usually a biopolymericcompound such as a protein e.g. a radiolabeled ligand for the receptor.After washing the vessel, interaction inhibition may be measured bydetermining the amount of detectable binding partner present in thevessel. Interaction inhibition occurs when binding of the bindingpartner is reduced greater than about 20%, greater than about 50%,greater than about 70%, greater than about 80%, or greater than about90% or 95% or more, as compared to a control assay that does not containantibody.

In the reaction inhibition assay, an enzyme may be bound to a solidsupport in a reaction vessel. Antibody is usually added to the reactionvessel followed by a substrate for the enzyme. In many embodiments, theproducts of the reaction between the enzyme and the substrate aredetectable, and, after a certain time, the reaction is usually stopped.After the reaction has been stopped, reaction inhibition may be measuredby determining the level of detectable reaction product present in thevessel. Reaction inhibition occurs when the rate of the reaction isreduced greater than about 20%, greater than about 50%, greater thanabout 70%, greater than about 80%, or greater than about 90% or 95% ormore, as compared to a control assay that does not contain antibody.

In Vivo Assays

In certain embodiments the antibodies are tested in vivo. In general,the method involves administering a subject monoclonal antibody to ananimal model for a disease or condition and determining the effect ofthe monoclonal antibody on the on the disease or condition of the modelanimal. In vivo assays of the invention include controls, where suitablecontrols include a sample in the absence of the monoclonal antibody.Generally a plurality of assay mixtures is run in parallel withdifferent antibody concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection.

Substituted Antibodies

The present invention provides substituted antibodies that aresubstituted by the method set forth above.

In general, a substituted antibody retains specificity for an antigen ascompared to a parent antibody, has substantial affinity (e.g. at least10⁷M⁻¹, at least 10⁸ M⁻¹, or at least 10⁹M⁻¹ to 10¹⁰ M⁻¹ or more) tothat antigen, and, if humanized, is usually less immunogenic in a humanhost, as compared to a parent antibody.

The level of immunogenicity of a humanized antibody as compared to aparent rabbit antibody in a human host may be determined by any of anumber of means, including administering to a single human host aformulation containing equimolar amounts of the two isolated antibodiesand measuring the immune response of the human host relative to each ofthe antibodies. Alternatively, the parent and modified antibodies areadministered separately to different human hosts and the immune responseof the hosts are measured. One suitable method for measuring the immuneresponse of the host relative to each of the antibodies is by ELISA(described in Ausubel, et al, Short Protocols in Molecular Biology, 3rded., Wiley & Sons, 1995, UNIT 11-4), where a suitable equal amount ofeach antibody is spotted into the wells of a microtitre plate, and theassay is performed polyclonal antiserum from the human host. In mostembodiments, a subject humanized antibody is about 10% less immunogenic,about 20% less immunogenic, about 30% less immunogenic, about 40% lessimmunogenic, about 50% less immunogenic, about 60% less immunogenic,about 80% less immunogenic, about 90% less immunogenic or even about 95%less immunogenic than an unmodified parent antibody.

Depending on the constant regions and other regions used, several typesof antibody that are known in the art may be made. As well as fulllength antibodies, antigen-binding fragments of antibodies may be madeby the subject methods. These fragments include, but are not limited to,Fab, Fab′ and F(ab)₂, Fd, single-chain Fvs (scFv), single-chainimmunoglobulins (e.g., wherein a heavy chain, or portion thereof, andlight chain, or portion thereof, are fused), disulfide-linked Fvs(sdFv), diabodies, triabodies, tetrabodies, scFv minibodies, Fabminibodies, and dimeric scFv and any other fragments comprising a V_(L)and a V_(H) domain in a conformation such that a specific antigenbinding region is formed. Antibody fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entire or partial of the following: a heavy chain constantdomain, or portion thereof, e.g., a CH1, CH2, CH3, transmembrane, and/orcytoplasmic domain, on the heavy chain, and a light chain constantdomain, e.g., a C_(kappa) or C_(lambda) domain, or portion thereof onthe light chain. Also included in the invention are any combinations ofvariable region(s) and CH1, CH2, CH3, C_(kappa), C_(lambda),transmembrane and cytoplasmic domains. By the term “antibody” is meantany type of antibody, including those listed above, in which the heavyand light chains have been, as explained above, naturally paired, i.e.,excluding so-called “phage-display” antibodies.

Nucleic Acids Encoding Substituted Antibodies

The invention further provides nucleic acids comprising a nucleotidesequence encoding a subject modified antibody, as well as portionsthereof, including a light or heavy chain, a light or heavy chainvariable domain, or a framework region of a light or heavy chainvariable domain. Subject nucleic acids are produced by a subject method.In many embodiments, the nucleic acid also comprises a coding sequencefor a constant domain, such as a constant domain of any human antibody.Nucleic acids encoding a human immunoglobulin leader peptide (e.g.MEFGLSWVFLVAILKGVQC, SEQ ID NO:53) may be engineered to allow thesecretion of the antibody chains.

Since the genetic code and recombinant techniques for manipulatingnucleic acid are known, and the amino acid sequences of the subjectantibodies may be obtained using the method described above, the designand production of nucleic acids encoding a substituted antibody is wellwithin the skill of an artisan. In certain embodiments, standardrecombinant DNA technology (Ausubel, et al, Short Protocols in MolecularBiology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., MolecularCloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,N.Y.) methods are used. For example, antibody coding sequences may beisolated from antibody-producing cells using any one or a combination ofa variety of recombinant methods that do not need to be describedherein. Subsequent substitution, deletion, and/or addition ofnucleotides in the nucleic acid sequence encoding a protein may also bedone use standard recombinant DNA techniques.

For example, site directed mutagenesis and subcloning may be used tointroduce/delete/substitute nucleic acid residues in a polynucleotideencoding an antibody. In other embodiments, PCR may be used. Nucleicacids encoding a polypeptide of interest may also be made by chemicalsynthesis entirely from oligonucleotides (e.g., Cello et al., Science(2002) 297:1016-8).

In certain embodiments, the codons of the nucleic acids encodingpolypeptides of interest are optimized for expression in cells of aparticular species, particularly a mammalian, e.g., human, species.

The invention further provides vectors (also referred to as“constructs”) comprising a subject nucleic acid. In many embodiments ofthe invention, the subject nucleic acid sequences will be expressed in ahost after the sequences have been operably linked to an expressioncontrol sequence, including, e.g. a promoter. The subject nucleic acidsare also typically placed in an expression vector that can replicate ina host cell either as an episome or as an integral part of the hostchromosomal DNA. Commonly, expression vectors will contain selectionmarkers, e.g., tetracycline or neomycin, to permit detection of thosecells transformed with the desired DNA sequences (see, e.g., U.S. Pat.No. 4,704,362, which is incorporated herein by reference). Vectors,including single and dual expression cassette vectors are well known inthe art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed.,Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Suitablevectors include viral vectors, plasmids, cosmids, artificial chromosomes(human artificial chromosomes, bacterial artificial chromosomes, yeastartificial chromosomes, etc.), mini-chromosomes, and the like.Retroviral, adenoviral and adeno-associated viral vectors may be used.

A variety of expression vectors are available to those in the art forpurposes of producing a polypeptide of interest in a cell. One suitablevector is pCMV, which used in certain embodiments. This vector wasdeposited with the American Type Culture Collection (ATCC) on Oct. 13,1998 (10801 University Blvd., Manassas, Va. 20110-2209 USA) under theprovisions of the Budapest Treaty for the International Recognition ofthe Deposit of Microorganisms for the Purpose of Patent Procedure. TheDNA was tested by the ATCC and determined to be viable. The ATCC hasassigned the following deposit number to pCMV: ATCC #203351.

The subject nucleic acids usually comprise an single open reading frameencoding a subject antibody, however, in certain embodiments, since thehost cell for expression of the polypeptide of interest may be aeukaryotic cell, e.g., a mammalian cell, such as a human cell, the openreading frame may be interrupted by introns. Subject nucleic acid aretypically part of a transcriptional unit which may contain, in additionto the subject nucleic acid 3′ and 5′ untranslated regions (UTRs) whichmay direct RNA stability, translational efficiency, etc. The subjectnucleic acid may also be part of an expression cassette which contains,in addition to the subject nucleic acid a promoter, which directs thetranscription and expression of a polypeptide of interest, and atranscriptional terminator.

Eukaryotic promoters can be any promoter that is functional in aeukaryotic, or any other, host cell, including viral promoters andpromoters derived from eukaryotic or prokaryotic genes. Exemplaryeukaryotic promoters include, but are not limited to, the following: thepromoter of the mouse metallothionein I gene sequence (Hamer et al., J.Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus(McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist etal., Nature (London) 290:304-310, 1981); the yeast gall gene sequencepromoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975,1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-59SS, 1984),the CMV promoter, the EF-1 promoter, Ecdysone-responsive promoter(s),tetracycline-responsive promoter, and the like. Viral promoters may beof particular interest as they are generally particularly strongpromoters. In certain embodiments, a promoter is used that is a promoterof the target pathogen. Promoters for use in the present invention areselected such that they are functional in the cell type (and/or animal)into which they are being introduced. In certain embodiments, thepromoter is a CMV promoter.

In certain embodiments, a subject vector may also provide for expressionof a selectable marker. Suitable vectors and selectable markers are wellknown in the art and discussed in Ausubel, et al, (Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al,(Molecular Cloning: A Laboratory Manual, Third Edition, (2001) ColdSpring Harbor, N.Y.). A variety of different genes have been employed asselectable markers, and the particular gene employed in the subjectvectors as a selectable marker is chosen primarily as a matter ofconvenience. Known selectable marker genes include: the thimydine kinasegene, the dihydrofolate reductase gene, the xanthine-guaninephosphoribosyl transferase gene, CAD, the adenosine deaminase gene, theasparagine synthetase gene, the antibiotic resistance genes, e.g. tetr,ampr, Cmr or cat, kanr or neor (aminoglycoside phosphotransferasegenes), the hygromycin B phosphotransferase gene, and the like.

The subject nucleic acids may also contain restriction sites, multiplecloning sites, primer binding sites, ligatable ends, recombination sitesetc., usually in order to facilitate the construction of a nucleic acidencoding a humanized rabbit antibody. In general, several methods areknown in the art for producing antibody-encoding nucleic acids,including those found in U.S. Pat. Nos. 6,180,370, 5,693,762, 4,816,397,5,693,761 and 5,530,101. One PCR method utilizes “overlapping extensionPCR” (Hayashi et al., Biotechniques. 1994: 312, 314-5) to createexpression cassettes for the heavy and light chain encoding nucleicacids. In this method multiple overlapping PCR reactions using the cDNAproduct obtained from the antibody producing cell and other appropriatenucleic acids as templates generates an expression cassette.

Methods for Producing Antibodies

In many embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cellincubated under conditions sufficient to induce expression of theencoded antibody.

Any cell suitable for expression of expression cassettes may be used asa host cell. For example, yeast, insect, plant, etc., cells. In manyembodiments, a mammalian host cell line that does not ordinarily produceantibodies is used, examples of which are as follows: monkey kidneycells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7,ATCC CRL 165 1); human embryonic kidney cells (HEK-293, Graham et al. J.Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl. Acad.Sci. (USA) 77:4216, (1980); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70);african green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658);and mouse L cells (ATCC CCL-1). Additional cell lines will becomeapparent to those of ordinary skill in the art. A wide variety of celllines are available from the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209.

Methods of introducing nucleic acids into cells are well known in theart. Suitable methods include electroporation, particle gun technology,calcium phosphate precipitation, direct microinjection, and the like.The choice of method is generally dependent on the type of cell beingtransformed and the circumstances under which the transformation istaking place (i.e. in vitro, ex vivo, or in vivo). A general discussionof these methods can be found in Ausubel, et al, Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons, 1995. In some embodimentslipofectamine and calcium mediated gene transfer technologies are used.

After the subject nucleic acids have been introduced into a cell, thecell is typically incubated, normally at 37° C., sometimes underselection, for a period of about 1-24 hours in order to allow for theexpression of the antibody. In most embodiment, the antibody istypically secreted into the supernatant of the media in which the cellis growing in.

In mammalian host cells, a number of viral-based expression systems maybe utilized to express a subject antibody. In cases where an adenovirusis used as an expression vector, the antibody coding sequence ofinterest may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing the antibody molecule ininfected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA81:355-359 (1984)). The efficiency of expression may be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544(1987)).

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that interact directly or indirectly with the antibodymolecule.

Once an antibody molecule of the invention has been produced, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In many embodiments, antibodies are secretedfrom the cell into culture medium and harvested from the culture medium.

Determining Binding Affinity of an Antibody

Once a modified antibody is produced, it may be tested for affinityusing any known method, such as: 1) competitive binding analysis using alabeled (radiolabeled or fluorescent labeled) parent antibody, amodified antibody and an antigen recognized by the parent antibody; 2)surface plasmon resonance using e.g. BIACore instrumentation to providethe binding characteristics of an antibody. Using this method antigensare immobilized on solid phase chips and the binding of antibodies inliquid phase are measured in a real-time manner; and 3) flow cytometry,for example, by using fluorescent activated cell sorting (FACS) analysisto study antibody binding to cell surface antigens; 4) ELISA; 5)equibrilium dialysis, or FACS. In this FACS method both transfectedcells and native cells expressing the antigen can be used to studyantibody binding. Methods for measuring binding affinity are generallydescribed in Harlow et al., Antibodies: A Laboratory Manual, FirstEdition (1988) Cold spring Harbor, N.Y.; Ausubel, et al, Short Protocolsin Molecular Biology, 3rd ed., Wiley & Sons, 1995).

If affinity analysis reveals a decrease in antibody binding for themodified antibody as compared to its parent antibody, “fine tuning” maybe performed to increase the affinity. One method of doing this is tosystematically change back each modified residues by site-directedmutagenesis. By expressing and analyzing these back mutant antibodies,one would predict the key residues that cannot be modified unlesswithout decreasing affinity.

Utility

An antibody produced by the instant methods finds use in diagnostics, inantibody imaging, and in treating diseases treatable by monoclonalantibody-based therapy. In particular, an antibody humanized by theinstant methods may be used for passive immunization or the removal ofunwanted cells or antigens, such as by complement mediated lysis orantibody mediated cytotoxicity (ADCC), all without substantial immunereactions (e.g., anaphylactic shock) associated with many priorantibodies. For example, the antibodies of the present invention may beused as a treatment for a disease where the surface of an unwanted cellspecifically expresses a protein recognized the antibody (e.g. HER2, orany other cancer-specific marker) or the antibodies may be used toneutralize an undesirable toxin, irritant or pathogen. Humanizedantibodies are particularly useful for the treatment of many types ofcancer, for example colon cancer, lung cancer, breast cancer prostatecancer, etc., where the cancers are associated with expression of aparticular cellular marker. Since most, if not all, disease-relatedcells and pathogens have molecular markers that are potential targetsfor antibodies, many diseases are potential indications for humanizedantibodies. These include autoimmune diseases where a particular type ofimmune cells attack self-antigens, such as insulin-dependent diabetesmellitus, systemic lupus erythematosus, pernicious anemia, allergy andrheumatoid arthritis; transplantation related immune activation, such asgraft rejection and graft-vs-host disease; other immune system diseasessuch as septic shock; infectious diseases, such as viral infection orbacteria infection; cardiovascular diseases such as thrombosis andneurological diseases such as Alzeimer's disease. An antibody ofparticular interest is one that modulates, i.e., reduces or increases asymptom of the animal model disease or condition by at least about 10%,at least about 20%, at least about 25%, at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 80%, at least about 90%, or more, whencompared to a control in the absence of the antibody. In general, amonoclonal antibody of interest will cause a subject animal to be moresimilar to an equivalent animal that is not suffering from the diseaseor condition. Monoclonal antibodies that have therapeutic value thathave been identified using the methods and compositions of the inventionare termed “therapeutic” antibodies.

Kits

Also provided by the subject invention are kits for practicing thesubject methods, as described above. The subject kits at least includeone or more of: a substituted antibody made according to the abovemethods, a nucleic acid encoding the same, or a cell containing thesame. The substituted antibody may be humanized. Other optionalcomponents of the kit include: restriction enzymes, control primers andplasmids; buffers; etc. The nucleic acids of the kit may also haverestrictions sites, multiple cloning sites, primer sites, etc tofacilitate their ligation to non-rabbit antibody CDR-encoding nucleicacids. The various components of the kit may be present in separatecontainers or certain compatible components may be precombined into asingle container, as desired.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Also provided by the subject invention is are kits including at least acomputer readable medium including programming as discussed above andinstructions. The instructions may include installation or setupdirections. The instructions may include directions for use of theinvention with options or combinations of options as described above. Incertain embodiments, the instructions include both types of information.

Providing the software and instructions as a kit may serve a number ofpurposes. The combination may be packaged and purchased as a means forproducing rabbit antibodies that are less immunogenic in a non-rabbithost than a parent antibody, or nucleotide sequences them.

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Identification of Variation Tolerant Amino Acids in anAnti-TNFα Rabbit Monoclonal Antibody

A rabbit was immunized with TNFα, the spleen of that rabbit was used tomake hybridoma cells, and hybridoma cells expressing anti-TNFαmonoclonal antibodies were isolated. cDNAs encoding the heavy and lightchains of those monoclonal antibodies were isolated from the isolatedcells, and sequenced. The polypeptides encoded by the cDNAs were alignedaccording to their structural features, and this alignment is shown inFIG. 4. FIG. 4 shows that two groups of related anti-TNFα, rabbitmonoclonal Abs were obtained. Antibodies 52, 63, and 115 belong to onegroup. Antibodies 1 and 204 belong to a different group. Positionsindicated by an asterisk (*) are non-variant positions, whereinpositions indicated by a period (.) or colon (:) are variant tolerantpositions. Many variation tolerant positions are within the CDRs.

FIG. 2 is a multiple sequence alignment of the H3 region of ten rabbitantibody sequences extracted from the Kabat database to illustrate theexpected variation in unrelated antibodies.

Example 2 Humanizing an Anti-TNFα Rabbit Monoclonal Antibody

The sequence of a rabbit anti-TNFα rabbit monoclonal antibody A52 isaligned with the most similar human germline antibody, L20, andvariation tolerant positions of the rabbit anti-TNFα rabbit monoclonalantibody are substituted with amino acids at the corresponding positionsof the L20 antibody to produce a humanized rabbit antibody (HZD). Thesubstituted amino acids are marked by stars. According to FIG. 4,position 31 (within a CDR) is a variation tolerant position because itis an N or an S. N was chosen since that is found in the human germlineantibody at that position. According to FIG. 4, position 48 (justoutside a CDR) is a variation tolerant position because it is an M or anI. I was chosen since that is found in the human germline antibody atthat position. According to FIG. 4, position 50 (within a CDR), is avariation tolerant position because it is an L or a V. This position wassubstituted with an A since A is the amino acid found in the humangermline antibody at that position. According to FIG. 4, position 70(within a framework region), is a variation tolerant position because itis an E or a Q. This position was substituted with a D because D isfound in the human germline antibody at this position. According to FIG.4, position 95B (within a CDR) is a variation tolerant position becauseit is a D or an N. This position was substituted with an N since N isless polar than N and therefore likely to be less immunogenic.

It is evident from the above results and discussion that the subjectinvention provides an important new means for making amino acids changesto an antibody. As such, the subject methods and systems find use in avariety of different applications, including research, agricultural,therapeutic and other applications. In particular, the inventionprovides a means for humanizing the antigen binding region (e.g., theCDR regions) of a non-human antibody. Accordingly, the present inventionrepresents a significant contribution to the art.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-36. (canceled)
 37. A method of altering the amino acid sequence of aparent antibody, comprising: a) identifying a variation tolerantposition in the amino acid sequence of said parent antibody by comparingthe amino acid sequence of said parent antibody to the amino acidsequence of a related antibody that is clonally related to said parentantibody, wherein said parent antibody and said related antibody arederived from an ancestral antibody via affinity maturation; and b)substituting the amino acid present at said variation tolerant positionwith a different amino acid to produce a modified antibody that bindsthe same antigen as said parent antibody but has an amino acid sequencethat is different to the amino acid sequence of the parent antibody. 38.The method of claim 37, wherein the amino acid of said variationtolerant position of said parent antibody is substituted by an aminoacid present at a corresponding position in a related antibody.
 39. Themethod of claim 37, wherein the amino acid of said variation tolerantposition of said parent antibody is substituted by an amino acid presentat a corresponding position in a human antibody.
 40. The method of claim37, wherein said substituting comprises making a conservative amino acidsubstitution.
 41. The method of claim 37, wherein said substitutingcomprises making a non-conservative amino acid substitution.
 42. Themethod of claim 37, wherein said method comprises identifying andsubstituting at least two variant tolerant positions in said parentantibody.
 43. The method of claim 37, further comprising testing saidmodified antibody for binding to said antigen.
 44. The method of claim37, further comprising testing said modified antibody for more specificbinding to said antigen relative to said parent antibody.
 45. The methodof claim 43, wherein said testing is done by ELISA.
 46. The method ofclaim 37, wherein said variation tolerant position is in a CDR of saidparent antibody.
 47. The method of claim 37, wherein said variationtolerant position is in a framework region of said parent antibody. 48.The method of claim 37, wherein said parent antibody and said relatedantibody are derived from an ancestral antibody via affinity maturationin vivo.
 49. The method of claim 37, wherein said parent antibody andsaid related antibody are rabbit or mouse antibodies.
 50. The method ofclaim 37, further comprising: introducing a nucleic acid encoding saidmodified antibody into a cell; culturing said cell to produce saidmodified antibody; and isolating said modified antibody from said cell.51. The method of claim 37, wherein: said animal is a rabbit or a mouse;and the amino acid of said variation tolerant position of said parentantibody is substituted by an amino acid present at a correspondingposition in a human antibody.